The utility of any gene therapy strategy is defined by its balance between safety and effectiveness. While virus-derived vectors offer exceptional potential to target and deliver DNA cargo with high efficiency into the target cell, viral strategies often suffer in their safety profiling. Recent viral gene therapy-related patient mortalities in clinical trials highlight some of the safety issues attributed to the use of viral gene transfer systems that include, but are not limited to, unwanted immune responses to viral capsid proteins, regeneration of virulent viruses, and insertional mutagenesis. In contrast, non-viral strategies based on naked, lipoplexed or polyplexed plasmid DNA (pDNA) vectors generally offer safer gene therapy, vaccine design, and drug delivery approaches. Plasmid DNA vectors are relatively easy to generate and store and offer tremendous design capacity.
Several major barriers need to be considered in order to develop non-viral gene delivery systems as a therapeutic product to be safely administered in vivo. A successful transgene delivery system depends on the entrance of the DNA vector into the mammalian host nucleus and expression of the encoded transgene(s). While simple in theory, several cellular barriers must be overcome in practice. Vectors must be bio- and immune-compatible and avoid degradation by serum nucleases and immune detection by phagocytes, while travelling in the extracellular surroundings. Plasma nucleases digest the unprotected DNA within just a few minutes, so DNA vectors need to rapidly cross the plasma membrane of target cells. This is further complicated by the fact that the plasma membrane is composed of dense lipoprotein barriers that intrinsically inhibit efficient DNA translocation. Strategies to overcome this barrier include complexing DNA vectors with synthetic nanoparticles to form a structure similar to the plasma membrane or receptor-mediated endocytosis; i.e. targeted liposomes. However, while non viral gene delivery techniques work toward efficiency of DNA delivery, they generally prove poor in the delivery of pDNA vectors to the nuclear compartment. Many techniques are currently being investigated to enhance levels of non-viral gene transfer by targeting vectors to the nucleus. These techniques include modification of plasmids with DNA nuclear targeting sequences (DTS), covalent linkage of nuclear localization signals (NLS) to the plasmid DNA constructs, and attachment of import receptors such as karyopherins, to vectors that promote uptake through the nuclear membrane pore complex. Modification of DNA with NLS-conjugates seems to result in highly efficient expression of linear DNA, but not circular DNA, in combination with liposomal delivery vectors. This difference may be attributed to charge per unit ratio of linear versus supercoiled circular DNA and provides yet another intriguing opportunity for lcc vectors.
In addition to the aforementioned challenges, conventional non-viral gene delivery approaches may lead to unwanted immunological responses and oncogenesis, imparted by the presence of bacterial genetic elements in pDNA constructs. These include prokaryotic origins of replication, antibiotic resistance genes, as well as high-frequency immunostimulatory CpG motifs that activate Toll-like receptors in mammalian hosts. In order to improve the immuno-compatibility and durability of pDNA vectors, a new generation of plasmid vector has been constructed that exploit the bacteriophage λ derived integrase (Int)-attP or P1-derived Cre-loxP site-specific recombination systems to generate mini plasmids. These “minicircles” provide safer minimized transgene vectors by removing unwanted prokaryotic elements, thus enhancing bio- and immuno-compatibility in the mammalian host. The smaller size compared to the parental plasmid backbone also confers improved extracellular and intracellular bioavailability leading to efficient gene delivery and hence, improved gene expression.
A second group of modified vectors offering great promise, are linear covalently closed (Ice) plasmid DNA vectors. Aside from the obvious topological differences, lcc double-stranded DNA molecules are torsion-free as they are not subject to gyrase-directed negative supercoiling, and as such possess the properties of linear DNA. However, lcc DNA is not subject to ExoV exonuclease activity in prokaryotes and serum nucleases in mammalian hosts due to covalent linkage of linear ends, preventing degradation of the pDNA vector. Lee DNA vectors have been constructed by various in vitro strategies including the capping of PCR products. Minimalistic immunogenic defined gene expression (MIDGE) vectors are generated by the digestion of both prokaryotic and eukaryotic backbones after isolation of plasmid from bacterial cells, followed by ligation of the therapeutic expression cassette into hairpin sequences for end-refilling. This technology has shown promising results in various applications including the development of a Leishmania DNA vaccine and a colon carcinoma treatment. MIDGE vectors have also demonstrated up to 17 fold improved transgene expression profile in vivo in some tissues, compared to conventional pDNA vectors. Thus, lcc DNA vectors may in fact outperform their circular counterparts with respect to expression efficiency and bioavailability. However, large-scale production of lcc vectors via existing multistep in vitro processes requires considerable time and financial cost.
E. coli phage N15 was the first discovered temperate phage that does not integrate into its bacterial host genome in its lysogenic (prophage) state and instead exists as a linear covalently closed (Icc)plasmid that is actively partitioned to daughter cells. The Icc conformation is conferred by the cleaving-joining activity of the protelomerase protein (Prokaryotic Telomerase), TelN (˜72 kDa), acting upon the 56 bp telRL target sequence that is entirely sufficient to confer TelN-mediated processing and linearization both in vivo and in vitro. Similarly, phage PY54, isolated from Yersinia enterocolitica, maintains its prophage as a linear, circularly permuted, and covalently closed plasmid with telomere hairpin ends and a genome size of 46 kbp. The paralogous minimal protelomerase target site of PY54 is a 42 bp perfect palindrome, that unlike N15 , only partially functions in vivo in the absence of adjacent sequences. The paralogue of the N15 TelN protelomerase, Tel, encodes a 77 kDa protein with observably identical function, able to process recombinant plasmids containing the pal, 42 bp palindromic target site. The tel gene possesses 60% sequence identity to telN and the active recombinases are similar in size (˜77 kDa). In addition, there is a partial homology between the 42 bp PY54 pal site and the 56 bp N15 telRL site, where the ten central palindromic nucleotides (5′-TACGCGCGTA-3′ (SEQ ID NO: 17)) are identical. Despite obvious similarities between the two phages they are evolutionary quite distant, where N15 is more closely related to λ than to PY54. Purified TelN was shown to process circular and supercoiled plasmid DNA containing the identified target site, telRL, to produce linear double-stranded DNA with covalently closed ends. The Icc and mini Icc pDNA vectors produced in vitro by recombinant TelN have been successfully applied in gene delivery experiments, and showed higher and more durable expression of the gene of interest in targeted human cells.
Given the foregoing, it would be desirable to further develop alternative vector systems that provide one or more advantages over existing vectors.